Center of Clinical Reproductive Medicine, First Affiliated Hospital, Jiangsu Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China.
Reprod Biol Endocrinol. 2010 Apr 2;8:32. doi: 10.1186/1477-7827-8-32.
Although many nanomaterials are being used in academia, industry and daily life, there is little understanding about the effects of nanoparticles on the reproductive health of vertebral animals, including human beings. An experimental study was therefore performed here to explore the effect of calcium phosphate nanoparticles on both steroid hormone production and apoptosis in human ovarian granulosa cells.
Calcium phosphate nanoparticles uptaking was evaluated by transmission electron microscopy (TEM). The cell cycle was assessed with propidium iodide-stained cells (distribution of cells in G0/G1, S, and G2/M phases) by flow cytometry. The pattern of cell death (necrosis and apoptosis) was analyzed by flow cytometry with annexin V-FITC/PI staining. The expression of mRNAs encoding P450scc, P450arom and StAR were determined by RT-PCR. Progesterone and estradiol levels were measured by radioimmunoassay.
TEM results confirmed that calcium phosphate nanoparticles could enter into granulosa cells, and distributed in the membranate compartments, including lysosome and mitochondria and intracellular vesicles. The increased percentage of cells in S phase when cultured with nanoparticles indicated that there was an arrest at the checkpoint from phase S-to-G2/M (from 6.28 +/- 1.55% to 11.18 +/- 1.73%, p < 0.05). The increased ratio of S/(G2/M) implied the inhibition of DNA synthesis and/or impairment in the transition of the S progression stage. The apoptosis rate of normal granulosa cells was 7.83 +/- 2.67%, the apoptotic rate increased to 16.53 +/- 5.56% (P < 0.05) after the cells were treated with 100 microM calcium phosphate nanoparticles for 48 hours. Treatment with calcium phosphate nanoparticles at concentrations of 10-100 microM didn't significantly change either the progesterone or estradiol levels in culture fluid, and the expression levels of mRNAs encoding P450scc, P450arom and StAR after 48 h and 72 h period of treatment.
Calcium phosphate nanoparticles interfered with cell cycle of cultured human ovarian granulosa cells thus increasing cell apoptosis. This pilot study suggested that effects of nanoparticles on ovarian function should be extensively investigated.
尽管许多纳米材料在学术界、工业界和日常生活中得到了广泛应用，但人们对纳米颗粒对包括人类在内的脊椎动物生殖健康的影响知之甚少。因此，本实验研究旨在探讨磷酸钙纳米颗粒对人卵巢颗粒细胞类固醇激素生成和细胞凋亡的影响。
通过透射电子显微镜（TEM）评估磷酸钙纳米颗粒的摄取情况。用碘化丙啶染色的细胞（细胞在 G0/G1、S 和 G2/M 期的分布）通过流式细胞术评估细胞周期。用 Annexin V-FITC/PI 染色通过流式细胞术分析细胞死亡（坏死和凋亡）的模式。通过 RT-PCR 测定编码 P450scc、P450arom 和 StAR 的 mRNA 的表达。用放射免疫法测定孕酮和雌二醇水平。
TEM 结果证实磷酸钙纳米颗粒可进入颗粒细胞，并分布在包括溶酶体和线粒体以及细胞内囊泡在内的膜性隔室内。与纳米颗粒共培养时 S 期细胞的百分比增加表明存在从 S 期到 G2/M 期（从 6.28±1.55%增加到 11.18±1.73%，p<0.05）的检查点阻滞。S/（G2/M）比值的增加意味着 DNA 合成受到抑制和/或 S 期进展阶段的转变受损。正常颗粒细胞的凋亡率为 7.83±2.67%，细胞用 100 μM 磷酸钙纳米颗粒处理 48 小时后，凋亡率增加到 16.53±5.56%（P<0.05）。用 10-100 μM 浓度的磷酸钙纳米颗粒处理不会显著改变培养液中的孕酮或雌二醇水平，也不会改变 48 和 72 小时处理后编码 P450scc、P450arom 和 StAR 的 mRNA 的表达水平。
磷酸钙纳米颗粒干扰培养的人卵巢颗粒细胞的细胞周期，从而增加细胞凋亡。这项初步研究表明，应广泛研究纳米颗粒对卵巢功能的影响。
